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Lecture DetailsEdit

Rod Devenish; Week 5 MED1011; Biochemistry

Lecture ContentEdit

PKU can be screened biochemically, is where phenylalanine hydroxylase is defective (tryptophan is present instead of arginine) which prevents conversion of phenylalanine to tyrosine. When babies with PKU consume protein, phenylalanine accumulates and causes brain damage. If detected early, a low protein diet will help.

DNA screening can be done at any time, pre-natally, can also identify heterozygote carriers. PCR can be used on 8 cell embryo before implantation to screen for disease before implantation. This is not routine and CVS (10th week of pregnancy) or amniocentesis (13-17th week) is more common.

Routine specific methods of testing are allele-specific cleavage, allele-specific oligonucleotide hybridisation, and allele specific PCR. Relies on knowledge of exact mutation.

Allele specific cleavage is shown when mutation affects a restriction site, fragments generated by cutting restriction enzyme has different lengths for normal and mutated DNA. Allele specific oligonucleotide hybridisation is when oligonucleotides are produced that will hybridise to either the normal or mutated gene. Probe can be flourescently labelled. Easier and faster than allele specific cleavage. Allele specific PCR uses two different primers for PCR, one for mutant and one for normal which differ at the last base. Only if the primer is a match will the product be generated. If both sequences are present (heterozygote) then both primers will give a result.

Infectious disease can be diagnosed by DNA probes or PCR. Template target sequence is required and specificity of primers mean the test is extremely sensitive. HIV test may take a while for enough viral antibodies to form, viral genome integrates into T lymphocytes. DNA from a blood sample can be analysed with PCR with tagged hybridisation probe.

DNA fingerprinting uses highly polymorphic areas of DNA. Two types of inherited sequence are used- single nucleotide polymorphisms (SNP), short tandem repeats (STR) which are repetitive sequences that lead to repeats, 8 different STR sequences can be used for unique patterns. DNA is cut with restriction enzymes to produce fragments, enzymes cut at sites that flank the repeat sequences. Fragments are then separated by electrophoresis and blotted onto a membrane. Pattern is revealed by hybridisation allowing comparisons to be made between samples. Can also be used to identify ethnic populations.

Cells have unique mRNA, DNA microarrays allow screening of thousands of sequences at the same time. There are two classes- arrays of cDNAs delivered by direct microspotting to a glass surface (chip), arrays of oligonucleotides can also be synthesised directly onto chip surface. These chips are used for expression screening (monitoring of RNA expression levels for the genome) and screening of DNA variation (oligonucleotide arrays used to assay mutations in known genes, catalogue human SNP markers).

Expression screening requires mRNA to be prepared from control and disease, mRNA is converted to cDNA, each cDNA population coloured with flourescent tag, these are placed in the recesses of a chip. Host cells are added. Intensity of tag represents abundance of transcript. Flouresence signals are evaluated using algorithms and allow visible discernment of pattern expression.

DNA chip technology used to determine gene expression signature of tumours, poor prognosis has a poor signature, determines treatment regimes. Pattern of spots provides information about the tumour.

Oligonucleotides can be produced matching all wildtype and single nucleotide substitution sequences in a gene, test DNA PCR'd, flourescently labelled, hybridised in competition with control labelled a different colour.

ReadingsEdit

Life (9th): 334-337, 325-326, 395-397, 163-164, 208-219Edit